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(11) | EP 0 397 775 B1 |
| (12) | EUROPEAN PATENT SPECIFICATION |
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APPARATUS AND METHOD FOR DETERMINATION OF A COMPONENT IN A SAMPLE VORRICHTUNG UND VERFAHREN ZUR BESTIMMUNG EINER KOMPONENTE EINER PROBE APPAREIL ET PROCEDE DE DETERMINATION D'UN COMPOSANT D'UN ECHANTILLON |
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| Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). |
Field of the invention
[0001] This invention relates to an apparatus useful in determining an analyte in a fluid sample. Especially it relates to an apparatus useful in determining an analyte using a four member or "quarternary complex".
BACKGROUND AND PRIOR ART
[0002] The formation of sandwiches of antigen and antibody and their use in immunoassays has been in use for over fifteen years. The art has seen two distinct trends in the field. The earliest trend was toward the formation of ternary complexes, i.e., complexes of the form - Ab₁-Ag-Ab₂*, where Ab₂* carries some label. The later trend is to multiple component systems, usually quaternary, but sometimes involving five or more components. The prior art discussion maintains this distinction.
I. Ternary Complex Formation
[0003] The patent literature contains many examples of inventions in this area. An early example of such an assay may be found in Schuurs, et al., U.S. Patent No. 3,654,090 (1972), which is useful not only as historical background, but for an understanding of some of the key facets of this field.
[0004] Schuurs, et al. teaches detection of an antigen using a solid phase bound antibody against one epitope, or binding site, of the antigen, as well as a soluble, enzyme labeled antibody which binds with a second portion of the antigen. The method disclosed in Schuurs, et al. involves determination of the enzyme label after the sandwich between bound antibody, antigen, and labeled antibody forms. This is accomplished either in the solid phase, or in the liquid phase, by addition of a substrate for the enzyme label. Usually, the enzyme-substrate reaction produces a color or change in color, which can be recognized in "yes-no" tests, or quantitated where the amount of substance present is to be determined.
[0005] Absent from Schuurs, et al. is any discussion of monoclonal antibodies or antibody fragments and this is not surprising since Schuurs, et al. was filed in 1968, and issued in 1972, i.e., much earlier than the breakthroughs in hybridoma technology which occurred following the development of the Köhler-Milstein method for producing monoclonal antibodies.
[0006] Schuurs, et al. received another patent in 1974, U.S. Patent No. 3,791,932, again directed to sandwich assays. This patent describes a so-called "forward" sandwich immunoassay. This type of assay calls for a specific order of steps, i.e., the sample being tested is first contacted with the insoluble binding partner and the reaction between these two is allowed to proceed to completion. The solid phase complexes are removed from the solution, and the second binding partner, containing an enzyme label, is then added to the solid phase. Following binding to the complex, the enzyme level is determined, following the standard techniques referred to, supra. Again, there is no mention of monoclonal antibodies or antibody fragments.
[0007] Ling, in U.S. Patent No. 3,867,517 (1975), taught that enzymes were not the only label which could be used in sandwich assays. This patent describes a forward sandwich assaying as the label a radioactive antibody. The radioactive label was ¹²⁵I, a standard radioisotope. Radiolabelling of antibodies is a standard technique, but assumes the presence of the proper amino acids in the antibody molecule for binding of the radioactive iodine. Otherwise, the label does not hold.
[0008] Schuurs, et al., received yet another patent in 1977, U.S. Patent No. 4,016,043. This patent claims to teach a simpler version of rudimentary sandwich assays. It teaches using an insoluble component of an antigen-antibody reaction and a labeled sample of the same component. This method assumes that the antigen being detected has two identical epitopic sites. Further, the use of two identical receptors precludes the use of "simultaneous" assays, which are discussed infra. The consequences of this is that the Schuurs ′043 assay can take as long as 60 hours to complete. In clinical or diagnostic laboratory, the large amount of time requires is unacceptable.
[0009] Piasio, et al., U.S. Patent No. 4,098,876 (1978) taught a "reverse" sandwich assay. This patent is important because it showed, first, that the component being determined could be bound to the soluble, labeled antibody first, and the immobilized antibody second. It was also an improvement in that a washing step was eliminated, which meant that time was saved in performing the assay. Piasio, et al. teach that their assay could, ideally, be completed in under one-half hour. This paradigmatic system was not realized in their examples, but the time was substantially less than the 60 hours for Schuurs, et al., discussed supra. A significant drawback of the method is that it requires enormous amounts of immobilized antibody.
[0010] Niswender, U.S. Patent No. 4,048,298 (1977), is actually not a sandwich assay, but shows an invention where an immobilized antibody was used to bind another antibody. This patent teaches an interesting variation on older competitive immunoassays. Niswender contacts a solid phase bound antibody with the sample being assayed as well as a second, radiolabeled antibody which binds to the first, but not to the component being determined. The effect of this is to allow the investigator to determine substance present by determining how much radiolabeled antibody binds to the solid phase.
[0011] This patent shows that antibodies can bind to other antibodies rather than just antigens. This property is important in more recent assays, some of which are discussed infra.
[0012] Schwarzberg, U.S. Patent No. 4,235,689 (1980) recognized that antibodies possess two distinct portions, the Fc portion, or "constant" region, and the Fab portion, which is the part of the antibody which binds to an epitopic site. Schwarzberg prepared complexes of labeled Fab fragments bound to a ligand, such as a polypeptide. This complex is then used in so-called "competitive" assays. No solid phase binding, or sandwich assays, are described.
[0013] Jeong, et al., U.S. Patent No. 4,244,940 (1981) teaches a "simultaneous" sandwich immunoassay. Such an assay requires an antigen with different epitopic sites, because two different antibodies or receptors must be used, for the reasons elaborated upon supra.
[0014] With Jeong, et al., it will be seen that by 1981 the state of the art in this field did teach forward, reverse, and simultaneous assay, always with ternary complexes (i.e., complexes of three species) being formed. The art had begun to see the use of Fab fragments as "linker" molecules (Schwarzberg), but they had not been used as an essential part of an immunoassay system, nor had monoclonal antibodies been used.
[0015] Both of these ideas were taught in patents which issued in 1983. David, et al., U.S. Patent No. 4,376,110 (1983), overcame a prejudice in the art that monoclonal antibodies were not "sticky" enough, i.e., possessed insufficient affinity for use in sandwich assays. David, et al., taught that all three forms of ternary sandwich assays could be performed with monoclonal antibodies, as long as they both had affinities of at least 10⁸ liters/mole. Moussebois, et al., in U.S. Patent No. 4,397,060 (1983), taught an agglutination assay could be performed using Fab fragments bound to a solid support. This patent shows, yet again, that Fab fragments were not being considered as partners of immunoassays, even though monoclonal antibodies themselves were now being used.
[0016] Gallati, et al., U.S. Patent No. 4,467,031 (1984) taught a specific sandwich assay, for determination of carcinoembryonic antigen (CEA). The key feature of this invention was the use of different salt concentrations to improve complex formation. It is a "forward" sandwich assay, as the term is defined herein, and discusses the possibility of two monoclonal antibodies being used in the assay. It will be seen that this, too, is a ternary complex, and that an Fab fragment is not being used.
[0017] Woods, et al., U.S. Patent No. 4,469,787 (1984) teaches a sandwich assay which requires the binding of a label to the Fc portion of a second antibody. The label is not directly attached to the second antibody, rather, Woods et al. assert invention in that the label is bound to the Fc portion of the antibody after the ternary complex is formed. This is done so as to prevent interference between the label and the immobilized first antibody.
[0018] U.S. Patent No. 4,486,530 (1984), which issued to David, et al., and is a continuation in part of U.S. Patent No. 4,376,110, discussed supra, again teaches ternary monoclonal antibody sandwiches and their detection. This patent adds to the art by showing that sandwich assays can be performed in homogeneous phase, i.e., without phase separation. This is performed by labeling the monoclonal antibody components of the ternary complexes with labels which do not react unless brought together by the "glue" of a multiepitopic antigen.
[0019] Carro, et al., U.S. Patent No. 4,522,922 (1985) combine sandwich assays with an older form of immunoassay, the so-called "precipitation" test. This invention teaches formation of a ternary sandwich, followed by addition of a precipitating agent to precipitate the complex out of solution. This is a radioimmunoassay, which employs polyclonal antisera.
[0020] The most recent patents in the field show modifications on the basic sandwich principle. Petska, in U.S. Patent No. 4,623,621 (1986), teaches that an oligomeric protein can be measured by using a solid phase bound monoclonal antibody which is specific for an epitope present once on the repeating protein portion of the molecule. After solid phase binding, a second sample of the same monoclonal antibody, only labeled, is bound. Again, a ternary complex is formed, only with whole antibodies, and simultaneous assaying is not possible.
II. Multiple Member Complex Formation
[0021] The earliest example of a quaternary system is exhibited by U.S. Patent No. 4,343,896, which issued to Wolters, et al. This patent which is based on a disclosure filed in 1976, teaches the solid phase bound complex Ab₁-Ab₂-Ag-Ab₃*. A crucial limitation in the Wolters patent is that Ab₂ and Ab₃* come from different animal species. The reason for this is because Ab₁ has to be directed against the constant region, i.e., "Fc" portion of Ab₂. All antibodies of a particular immunological class which come from the same animal species will have identical Fc portions. If Ab₂ and Ab₃ were from the same animal species, the art taught that not only would Ab₁-Ab₂-Ag-Ab₃* but one one would also obtain Ab₁-Ab₃*, both of which would bind to the solid phase, causing interference and incorrect results.
[0022] Axen, et al., U.S. Patent No. 4,469,796 (1984) teaches that more than three components may be involved in an immune reaction, but the only four part complex taught is a solid phase bound complex of Ag-Ab₁-Ab₂-Ab₃*. It is is a solid phase bound complex of Ag-Ab₁-Ab₂-Ab₃*. It is noteworthy that in the description of reactants given at column 1, lines 41-60, Axen, et al. never mentions Fab fragments.
[0023] Tanswell, et al., U.S. Patent No. 4,624,930 (1986) teaches four component complexes wherein a first and third receptor in solution bind to the antigen while a second solid phase antibody binds to the first antibody. Tanswell's teaching is generic to the use of a double antibody system and it does not specifically disclose monoclonal antibodies.
[0024] Forrest, et al., U.S. Patent No. 4,659,678 (1987) goes beyond the four part binding discussed supra, and actually forms a pentavalent complex of antibody-hapten-antibody-antigen-antibody. The tail end of the complex is a radioactively labeled antibody. At least one antibody must be a monoclonal antibody.
[0025] Forrest, et al. detail at some length the advantages and disadvantages of multi-member complex forming assays. The solution to the problems set forth at, e.g., column 2, lines 1-5, is to use a solid phase bound mAb, to bind a complex of Ab-Ag-Fab*. The only time a solid phase bound mAb is used to bind the complex mAb₂-Ag-Fab*, however, Forrest requires that the mAb₂ be bound to another antigen, so that the solid phase complex mAb₁-Ag₂-mAb₂-Ag₁-Fab* is formed. It must be understood in this context, however, that "Ag₂" actually stands for a linking agent, as mAb₂ cannot possess two Ag binding sites.
Multilayer analysis elements
[0026] Multilayer analysis elements for immunological determinations with blocking layers are described in U.S. patent Nos. 4,459, 358 and 4, 613, 567. These blocking layers serve only for the visual discrimination for the detection in two different layers and have no influence in the diffusion of the test solution:
[0027] US-P-4,459,358 describes a multilayer element with a reaction layer, an insulating layer and a registration layer. The only task of the insulating layer is to prevent the detection of the detectable species in the reaction layer and to limit the detection to those species, which have diffused to the registration layer.
[0028] US-P-4, 613, 567 describes a multilayer element with a first detection layer, a blocking layer and a second detection layer. The blocking layer includes pigments as blocking agents for light. Thus it is possible to measure on both detection layers.
[0029] A two-layer element which includes a medium for retaining a sample solution for a certain time period is described in US-P-4 587, 102. The element comprises a detection layer and a reaction layer, whereby the reaction layer comprises a fibrous porous medium having water retention capability.
SUMMARY OF THE INVENTION
[0030] This application is directed to an apparatus which can be used in immunoassays such as immunoenzymometric assays, competitive assays and displacement assays.
[0031] Especially it can be used in a method for determining of a quarternary complex between a solid phase bound antibody which binds to the Fc portion of a monoclonal antibody but not to the Fab portion, a whole monoclonal antibody which binds to the analyte, the analyte itself and a labelled Fab fragment of a monoclonal antibody.
[0032] Thus the invention is directed to an apparatus for determining an analyte in a sample, comprising:
(a) a first zone containing a labeled sample of one of (i) the analyte to be determined, (ii) an analogue of the analyte, and (iii) a receptor which specifically binds to said analyte,
(b) a second zone which is in at least partial fluid contact with said first zone and is adapted for receiving fluid from said first zone, said second zone containing a solid phase bound receptor which binds to one of (i) both sample analyte and labeled analyte, (ii) both sample analyte and labeled analyte analogue, or (iii) labeled receptor not bound to sample analyte,
(c) a third zone which contains a reactive component, which reactive component combines with the label on said labeled analyte, labeled analyte analogue or labeled receptor to form a detectable moiety, said third zone being separated from said second zone by a means capable of being rendered fluid permeable, said means positioned so as to prevent passage of said reactive component from said third zone and into said second zone prior to completion of the reaction between said solid phase bound receptor and unbound label carrying sample, and
(d) a fourth zone in at least partial fluid contact with said second zone and adapted for receiving fluid from said second zone.
[0033] Another embodiment of the invention is an apparatus for determining an analyte in a fluid sample comprising:
(a) a first zone containing a sample of a plurality of different, non-solid phase bound receptors which specifically bind to said analyte, one of said receptors carrying a label and a second one not,
(b) a second zone which is in at least partial fluid contact with said first zone and is adapted from receiving fluid from said first zone, said second zone containing a solid phase receptor which binds to the first zone receptor which does not carry a label and specifically not binding to said labeled receptor,
(c) a third zone which contains a reactive component, which reactive component combines with the label on said labeled analyte, labeled analyte analogue or labeled receptor to form a detectable moiety, said third zone being separated from said second zone by a means capable of being rendered fluid permeable, said means positioned so as to prevent passage of said reactive component from said third zone and into said second zone prior to completion of the reaction between said solid phase bound receptor and unbound label carrying sample, and
(d) a fourth zone in at least partial fluid contact with said second zone and adapted for receiving fluid from said second zone.
[0034] How these and other aspects of the invention are achieved will be seen upon review of the disclosure which follows.
BRIEF DESCRIPTION OF THE FIGURES
[0037] Figure 3 shows another embodiment of the invention referred to as the "Delayed Physical Application System".
[0038] Figure 4 provides an embodiment of the invention referred to as the "delayed diffusion application system".
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0042] Referring now to Figure 1 a test strip 11 provided by this invention is shown. A stable carrier foil 12 is provided, which gives support to the entire apparatus. Optional sponge 13 is shown, which may contain, e.g., a buffer or other reagents useful in preparing the sample for analysis. Sample may be applied to first zone 14, by, e.g., pipette, and the sponge may be dipped directly into a liquid, or liquid may be applied directly by, e.g., a pipette.
[0043] The "first zone" is shown at 14, and contains at least one of the analyte to be determined, an analogue of this analyte, or a non-solid phase bound receptor which binds to the analyte being determined. Various substances are possible. Although it will frequently be the case that the analyte being determined is an antigen, such as a viral protein, a drug or drug residue, and so forth, other substances may be determined, especially if the sample being analyzed is not a biological fluid. The first zone 14 may also contain, in the case of a sandwich assay the two monoclonal antibodies from the same species which bind to the analyte being determined. When a sandwich assay is being performed, one of the two monoclonal antibodies contained in first zone 14 will carry a label, such as an enzyme.
[0044] The "third zone" 15 contains a substrate for the label carried by first zone 14. This substrate can be, e.g., a substrate acted upon by the enzyme, such as a beta galactoside when the enzyme is beta galactosidase. It may be a substance which is necessary for the label to function. For example, the substrate of the third zone may be a substance which combines with the label of the first zone to form a fluorescing moiety, or a functioning molecule. For example, the label and substrate may be halves of a complete enzyme which do not possess catalytic activity until brought together.
[0045] The first and third zones must be kept separate from each other, so that premature reaction between label and substrate does not occur. This is achieved via the blocking means 16, positioned between first and third zones 14 and 15. This blocking means need not be made of any particular material, as long as it prevents diffusion between the zones in 14 and 15 until the sample has entered the second zone.
[0046] The second zone 17 contains a solid phase bound receptor which binds to any of the reagent in first zone 14 which does not react with analyte from the sample, or when a sandwich assay is being performed, this second zone contains a solid phase bound receptor which binds only to the Fc portion of a monoclonal antibody.
[0047] An alternate construction divides this second zone into two portions, one of which is Fc specific, and the other which is not. In this case, formation of a signal, of course, is related to whether the quarternary complex formed, or did not. The non-specific matrix bound antibody thus serves as a negative control.
[0048] It is to be noted that the first zone 14 and second zone 17 must be in at least partial fluid contact with each other. Second zone 17 and third zone 15 may be in fluid contact, but need not be. The embodiment in Figure 1 actually shows no fluid contact between the second and third zones, because of the presence of barrier foil 18. This barrier foil serves to retard the passage of substrate from the third zone into the second zone. This permits whatever reactions are to occur between the solid phase bound component and the unreacted reagent of the first zone or sandwiches of mAb-Ag-Fab* to occur without premature formation of signal. As the substrate must, eventually enter the second zone, barrier 18 is made of fluid permeable material, preferably a polyvinyl alcohol, or material which, via contact with a surfactant or surface active agent, is made fluid permeable.
[0049] Fourth zone 19 is in contact with second zone 17, and receives excess sample and reagents therefrom. It acts as a "waste receptacle" for the device as a whole.
[0050] An optional cover slip 20 is provided as well. This gives additional stability to the device.
[0051] In Figure 2 a modification of the device of Figure 1 is shown. In device 21, all components are the same as in device 11, except it will be noted that sponge 13 now contacts first zone 14 directly, and does not contact third zone 15 directly. Rather, there is partial fluid contact between the first and third zones. Premature contact of label and substrate is avoided by positioning these at, e.g., the thatched positions 22 and 23, which are separated from each other by barrier 16.
[0052] Figure 3 shows an embodiment where the optional sponge 13 of Figures 1 and 2 is not used. Here, the device 31 contains first zone 32, to which sample is added directly. First zone 32 functions as does first zone 14 in Figures 1 and 2. It is in partial fluid contact with second zone 33, which, of course, functions in the same way as does the second zone 17 of Figures 1 and 2. A key distinction between the device of Figure 3 and that of Figures 1 and 2 is the placement and construction of the third zone, which contains the substrate. As will be seen by reference to Figure 3, the third zone, containing portions 34 and 35, is below the second zone and is in fluid contact therewith. The third zone contains substrate in region 35, and is supported by layer 34.
[0053] When layer 34 receives fluid which has migrated through component 43, it brings substrate 35 into contact with the second zone. Blocking layer 44 prevents fluid contact between zone 1 and component 43. Component 34 may be, for instance, a compressed sponge which swells when contacted by fluid.
[0054] In this configuration, premature reaction of label and substrate is not an issue, because by the time fluid reaches the third zone and releases the substrate, any reaction between the labeled reactant of the first zone and the solid phase bound reactant of the third zone has already taken place. The substrate diffuses into the second zone, where the detectable moiety is formed. Excess sample and reagents are carried into fourth zone 19, as in the embodiment of Figure 1, and the whole device is again held together by carrier foil 12.
[0055] An optional feature presented by this device is the covering means 36. The covering means allows for more precise observation of the reaction going on in the test strip. Generally, this covering means permits only selective viewing by providing viewing means or "windows" at various positions. Only one viewing means is actually necessary, and this should be over the second zone 33, so that formation of detectable moiety can be observed there. If the covering means 36 contains additional viewing means over fourth zone 19 one can observe reaction between substrate and labeled binding partner, e.g., or unreacted labeled Fab fragment. Also, if covering means 36 is adapted for use in, e.g., the device of Figures 1 and 2, a viewing means can be provided at a point where zones 1 and 3 meet. This allows the investigator to determine if premature mixing of label and substrate has occurred. The covering means can be made of various materials, including foils. It can also be an injection molded lid or cover which is part of an injection molded case or container means.
[0056] Figure 4 differs from the device of Figure 3, in that protective layer 37 covers substrate 35 in the third zone, and substrate diffusion into zone 2 is initiated by fluid from zone 2 penetrating protective layer 37. This gives greater assurance that premature mixing of substrate will not take place. The covering means 36, which, it has been pointed out, is optional, is not included in this embodiment, although it could have been.
[0057] Figure 5, the "loop embodiment" depicts the embodiment of the device where sponge 13 is used for sample application, as in Figure 2, supra. The sample passes into the first zone 14, where the reaction between, e.g., analyte and binding partner or mAb, Fab*, and Ag takes place. The whole content of first zone 14 passes to second zone 17, where either unreacted labeled substance or sandwiches are picked up by the solid phase bound reactant situated here. Anything not bound in second zone 17 is carried via means 38 through 39, which retains any label. The excess portion of the fluid sample enters waste 19 but, rather than being held here, the configuration of the device is such that the sample is forced into third zone 15, which contains the label substrate. As the configuration forces passage into third zone 15, it also precludes passage back to 19. Via means 52, the substrate containing material now passes through barrier 53 back into second zone 17, where reaction of solid phase bound labeled reactant and substrate takes place. Barrier 53 is selected so that while sample can pass through it from means 52, it cannot pass up from second zone 17.
[0058] Figure 6 shows the "integral matrix" embodiment of the device. In this embodiment, the second and third zones essentially become one in matrix 42. The substrate is incorporated into this matrix by means which may include, but are not limited to, encapsulation. The combining of the two zones in one matrix requires that the substrate not be released until such time as the labeled reactant from first zone 14 has reacted with solid phase bound material contained herein.
[0059] The final pictured embodiment in Figure 7 shows device 71. Here, all of the depicted elements are as in Figures 1 and 2, except that in this embodiment the third zone containing the substrate 15 is separated from second zone 17. Only by applying an external force to 15 can be substrate be brought into contact with the solid phase bound label.
[0060] Various assays may be performed in any and all of the preferred embodiments shown in Figures 1-7. For purposes of illustration, the mechanics of a different assay using the device of Figures 1, 4, and 5 are set out, although it will be clear to the skilled artisan that any and all of these may be adapted for use in any of the devices.
[0061] In performing a test for the presence of thyroxin (also called T4) in blood, e.g., a sample is applied to first zone 14 of the device of Figure 1. Tap water is applied to sponge 13 and migrates into third zone 15. The first zone contains T4 specific antibodies carrying the enzyme label horseradish peroxidase, while zone 15 contains any of the standard horseradish peroxidase substrates, such as orthophenylenediamine. The sample begins moving toward second zone 17, which contains, in solid phase bound and immobilized form, either T4 itself or related molecule T3. In moving through zone 14, any T4 in the sample has reacted with the horseradish peroxidase labeled T4 specific antibodies to form complexes. These, together with uncomplexed antibodies wash into second zone 17 ahead of the fluid which travelled through zone 15. The differential diffusion occurs because of the barrier 18.
[0062] While barrier 18 is dissolving, any uncomplexed antibody reacts with the solid phase bound T3 or T4 in the second zone 17, and the previously formed T4-antibody complex passes into waste zone 19. Substrate for horseradish peroxidase now passes into second zone 17, where it reacts with the enzyme immobilized on the solid phase. This produces a quantifiable signal, as will be recognized by those skilled in the art. The amount of enzyme caught by the solid phase is a measure of how much T3 or T4 was in the sample.
[0063] Similarly, one may perform a sandwich assay for, e.g., carcinoembryonic antigen (CEA), a multiepitopic substance, using the device of Figure 4. In such a test, first zone 32 contains both mouse-anti-human CEA monoclonal antibodies, and mouse-anti-human CEA monoclonal antibody Fab fragments labeled with beta galactosidase. Upon contact of first zone 32 with the sample, a sandwich forms between the whole antibody (MAb), the CEA (Ag) and the fragment (Fab*). This mAb-Ag-Fab* sandwich, together with unreacted mAb and Fab* pass into second zone 33, which contains, bound and immobilized to a solid phase, a sheep-antimouse Fc specific antibody. This solid phase binds both the sandwich described supra, as well as any excess mAb. As Fab* contains no Fc portion, however, this is not bound, and passes into the waste zone. Meanwhile, some of the sample has released the substrate resorufin beta galactopyranoside, which moves into the second zone 33. This substrate reacts with the Fab* fragments bound in this region, giving an indication of the presence and amount of CEA in the sample.
[0064] Using the device of Figure 5, one can perform a competitive assay for determining if a subject has been exposed to the HIV virus. This type of test assays for antibody rather than antigen, so it shows that, for purposes of this invention, these are equivalent.
[0065] Antibody to gp120 of HIV which is conjugated to an enzyme, such as a peroxidase, is incorporated into first zone 14 of device 51. A serum sample which may contain antibodies to HIV is introduced at sponge 13, and diffuses into 14. The mixture of sample and conjugate passes into second zone 17, which contains, immobilized in solid phase, HIV gpI20 sufficient to bind all of the labeled IgG if there is no other antibody present. Unbound conjugate will pass via means 38 into trap 39, which removes any free label from the sample. The remaining solution passes through third zone 15, releasing substrate, which passes via one way barrier into the matrix, where it reacts with bound label. There is an indirect correlation - i.e., the more label which bound, the less antibody there was in the sample, and vice versa.
[0066] Different materials may be used in each facet of the invention. As receptors, while antibodies are preferred, additional materials such as protein A, and biotin-avidin complexes, among others, can be used.
[0067] The immobilized receptor which forms the fourth part of the quaternary complex may be any of the materials listed supra, as long as it binds the first monoclonal antibody and does not bind monoclonal antibody fragments. Especially preferred are antibodies which bind to the Fc portion of other antibodies, but do not bind fragments.
[0068] When an antibody is used as the solid phase, a monoclonal antibody is preferred, although polyclonal antisera can also be used. The species in which the solid phase bound antibodies is generated is not important as long as there is no cross reactivity between the first receptor and the monoclonal antibody Fab fragment. The monoclonal antibody which binds to the antigen and the monoclonal antibody Fab fragment do derive from the same species, however.
[0069] The label used on the Fab fragment may be any of the conventional labels used in immunoassays, but especially preferred are enzymes which react with their substrates to form colored substrates. Examples of such enzymes are beta galactosidase, horseradish peroxidase, alkaline phosphatase urease and amylase, although it will be recognized that these are only examples and are not to be read as limits on what enzymes can be used. It will be clear to the skilled artisan, that when avidin is the matrix bound receptor, a biotinylated monoclonal antibody can be used. When this is the case, the labeled component need not be a Fab fragment, but can be a whole mAb.
[0070] The positioning of the labeled Fab fragment or mAb and first unlabeled or biotin/avidin labeled antibody in the first zone is not a critical feature of the invention. These can be positioned so that the sample reaches one before the other, or so that there is simultaneous contact.
[0071] The material of which the device is constructed can include many different items. Of course, the various zones must be absorptive of liquids and possess good capillarity. Examples of such materials are bibulous paper, nitrocellulose paper, sponges, and other absorptive materials. These may be fibrous or not, and the different zones can be composed of different materials possessing different degrees of capillarity, absorption, and so forth.
[0072] The receptor is immobilized via any of the standard means known in the art for immobilizing such receptors, e.g., to a solid support, such as by fixing with cyanogen bromide.
[0073] As mentioned supra, when the barriers are used in the apparatus, they must be chosen so that they permit fluid passage. Inert polymers are preferred, and especially preferred is polyvinyl alcohol (PVA). Other suitable materials will be evident to the skilled artisan.
[0074] When the cover means is used, its openings must be open, transparent or translucent. One preferred material for this is transparent mylar, while the rest of the cover can comprise suitably sturdy material, such as metallic foil may or may not be covered with a transparent material.
[0075] The additional features of the invention, such as the support and the impermeable barrier between the first zone and the substrate zone comprise conventional materials known to the art.
[0076] The following example illustrates the operation of the invention, but is not to be read as any limitative of the preceding discussion.
Example
[0078] A piece of 4210 paper (Firma Kalff) was cut into a strip 2.6 cm long and .6 cm wide (first zone). One end was impregnated with 10 µl PBS buffer (pH 7.0, 1% BSA, .1% Tween 20), and its center portion was impregnated with 7.5 µl of a solution containing 20 U/ml of a conjugate of an Fab portion of a monoclonal antibody against hCG and beta galactosidase. The monoclonal antibody fragment had no cross reactivity against luteinizing hormone. This portion was also impregnated with 75 µl of a 100 µg/ml solution of a monoclonal antibody against the beta chain of hCG. The end of the strip opposite the buffer impregnated end was impregnated with 10 µl of an aqueous solution of 5% polyvinyl alcohol. The resulting strip overlapped .5 mm of a strip of 3512 paper from Schleicher & Schull (second zone) which was 1 cm long and .6 cm wide. This paper had been activated using cyanogen bromide, and a sheep antibody against the Fc portion of mouse antibodies was fixed thereto. This strip overlapped .5 mm of a 5 cm long and .6 cm wide strip of D28 paper (Whatman), impregnated with 150 µl of an aqueous solution of 18% polyvinyl alcohol (waste zone). The three strips, overlapped as indicated to form a continuous strip, were mounted on a 10 cm long, .6 cm wide strip of polystyrene using adhesive tape. The strips thus produced were dipped, one each, into urine samples calibrated as containing 0, 100, 250, and 500 mIU/ml hCG. After 5 minutes, each strip was dipped into a solution of .8 mmol resorufin beta-galactopyranoside in 100 mmol Hepes buffer (pH 7.5), and allowed to develop for 5 minutes. All strips dipped into hCG containing urine exhibited bright fuchsia color at the second and waste zones, while the strip dipped in the sample containing no hCG was yellow in the second zone and fuschia in the third zone. The change in color is indicative of the action of beta galactosidase on the resorufin beta galactopyranoside in the second zone and waste zone.
[0079] The foregoing example, it will be seen, could be modified very easily by, e.g., having the resorufin beta galactopyranoside impregnated into a separate zone in the manner described supra, and the development of the color change could be observed through a covering means as has also already been described.
(a) a first zone (14, 32) containing a labeled sample of one of (i) the analyte to be determined, (ii) an analogue of the analyte, and (iii) a receptor which specifically binds to said analyte,
(b) a second zone (17, 33) which is in at least partial fluid contact with said first zone and is adapted for receiving fluid from said first zone (14, 32), said second zone (17, 33) containing a solid phase bound receptor which binds to one of (i) both sample analyte and labeled analyte, (ii) both sample analyte and labeled analyte analogue, or (iii) labeled receptor not bound to sample analyte,
(c) a third zone (15, 35) which contains a reactive component, which reactive component combines with the label on said labeled analyte, labeled analyte analogue or labeled receptor to form a detectable moiety, said third zone (15, 35) being separated from said second zone (17, 33) by a means (18, 44) capable of being rendered fluid permeable, said means positioned so as to prevent passage of said reactive component from said third zone (15, 35) and into said second zone (17, 33) prior to completion of the reaction between said solid phase bound receptor and unbound label carrying sample, and
(d) a fourth zone (19) in at least partial fluid contact with said second zone (17, 35) and adapted for receiving fluid from said second zone (17,33).
(a) a first zone (14, 32) containing a sample of a plurality of different, non-solid phase bound receptors which specifically bind to said analyte, one of said receptors carrying a label and a second one not,
(b) a second zone (17, 33) which is in at least partial fluid contact with said first zone (14, 32) and is adapted from receiving fluid from said first zone (14, 32), said second zone (17, 33) containing a solid phase receptor which binds to the first zone receptor which does not carry a label and specifically not binding to said labeled receptor,
(c) a third zone (15, 35) which contains a reactive component, which reactive component combines with the label on said labeled analyte, labeled analyte analogue or labeled receptor to form a detectable moiety, said third zone (15, 35) being separated from said second zone (17, 33) by a means (18, 44) capable of being rendered fluid permeable, said means positioned so as to prevent passage of said reactive component from said third zone (15, 35) and into said second zone (17, 33) prior to completion of the reaction between said solid phase bound receptor and unbound label carrying sample, and
(d) a fourth zone (19) in at least partial fluid contact with said second zone (17, 33) and adapted for receiving fluid from said second zone (17, 33)
(a) a first zone (14, 32) containing a labeled sample of one of (i) the analyte to be determined, (ii) an analogue of the analyte, and (iii) a receptor which specifically binds to said analyte,
(b) a second zone (17, 33) which is in at least partial fluid contact with said first zone and is adapted for receiving fluid from said first zone, (14, 32) said second zone (17, 33) containing a solid phase bound receptor which binds to one of (i) both sample analyte and labeled analyte, (ii) both sample analyte and labeled analyte analogue, or (iii) labeled receptor not bound to sample analyte,
(c) a third zone (15, 35) which contains a reactive component, which reactive component
combines with the label on said labeled analyte, labeled analyte analogue or labeled
receptor to form a detectable moiety, said third zone (15, 35) being separated from
said second zone (17, 33) by a gap or air space so as to prevent passage of said reactive
component from said third zone into said second zone prior to completion of the reaction
between said solid phase bound receptor and unbound label carrying sample, wherein
said third zone (15, 35) moves relative to said second zone (17, 33) to create
fluid contact and thereby initiate transfer of the reactive component to said second
zone, and
(d) a fourth zone (19) in at least partial fluid contact with said second zone (17, 33) and adapted for receiving fluid from said second zone (17, 33).
(a) a first zone (14, 32) containing a sample of a plurality of different, non-solid phase bound receptors which specifically bind to said analyte, one of said receptors carrying a label and a second one not,
(b) a second zone (17, 33) which is in at least partial fluid contact with said first zone (14, 32) and is adapted from receiving fluid from said first zone (14, 32) said second zone (17, 33) containing a solid phase receptor which binds to the first zone receptor which does not carry a label and specifically not binding to said labeled receptor,
(c) a third zone (15, 35) which contains a reactive component, which reactive component combines with the label on said labeled analyte, labeled analyte analogue or labeled receptor to form a detectable moiety, said third zone (15, 35) being separated from said second zone (17, 33) by a gap or air space so as to prevent passage of said reactive component from said third zone (15, 35) into said second zone (17, 33) prior to completion of the reaction between said solid phase bound receptor and unbound label carrying sample, wherein said third zone (15, 35) moves relative to said second zone (17, 33) to create fluid contact and thereby initiate transfer of the reactive component to said second zone (17, 33) and
(d) a fourth zone (19) in at least partial fluid contact with said second zone (17, 33) and adapted for receiving fluid from said second zone (17, 33).
(a) a first zone (14, 32) containing a labeled sample of one of (i) the analyte to be determined, (ii) an analogue of the analyte, and (iii) a receptor which sepcifically binds to said analyte,
(b) a second zone 42) which is in at least partial fluid contact with said first zone and adapted for receiving fluid from said first zone (14, 32), said second zone (42) containing both (1) a solid phase bound receptor which binds to one of (a) both the analyte to be determined an labeled analyte (b) both the analyte and labeled analyte analogue, or (c) labeled receptor which is not bound to the analyte to be determined, (2) a reactive component, which reactive component combines with said label to form a detectable moiety and (3) means for delaying combination of label and reactive component until reaction of the solid phase bound receptor with the unbound label carrying sample is substantially complete, and
(c) a third zone (19) in at least partial fluid contact with said second zone (42) and adapted for receiving fluid from said second zone (42).
(a) a first zone (14, 32) containing a sample of a plurality of different, non-solid phase bound receptors which specifically bind to said analyte, one of said receptors carrying a label and a second one not,
(b) a second zone (42) which is in at least partial fluid contact with said first zone and adapted for receiving fluid from said first zone (14, 32), said second zone containing both (1) a solid phase bound receptor which binds to one of (a) both the analyte to be determined and labeled analyte (b) both the analyte and labeled analyte analogue, or (c) labeled receptor which is not bound to the analyte to be determined, (2) a reactive component, which reactive component combines with said label to form a detectable moiety and (3) means for delaying combination of label and reactive component until reaction of the solid phase bound receptor with the unbound label carrying sample is substantially complete, and
(c) a third zone (19) in at least partial fluid contact with said second zone (42) and adapted for receiving fluid from second zone (42).
(a) eine erste Zone (14, 32), welche eine markierte Probe der einen (i) zu bestimmenden Komponente, (ii) eines Analoges der Komponente oder (iii) eines Rezeptors, welcher sich an besagte Komponente spezifisch bindet, enthält,
(b) eine zweite Zone (17, 33), welche in mindestens teilweisem Fluidkontakt mit besagter erster Zone steht und zur Fluidaufnahme aus besagter erster Zone (14, 32) angepasst ist, wobei besagte zweite Zone (17, 33) einen an eine feste Phase gebundenen Rezeptor enthält, der sich an eines der folgenden bindet: (i) sowohl die Komponente der Probe als auch die markierte Komponente, (ii) sowohl die Komponente der Probe als auch das markierte Analoge der Komponente, oder (iii) den an die Komponente der Probe nicht gebundenen, markierten Rezeptor,
(c) eine dritte Zone (15, 35), die eine reaktionsfähige Komponente enthält, welche reaktionsfähige Komponente sich mit dem Markierer auf besagter markierter Komponente, auf dem markierten Analogen der Komponente oder auf dem markierten Rezeptor unter Bildung eines detektierbaren Komplexes verbindet, wobei besagte dritte Zone (15, 35) von besagter zweiter Zone (17, 33) durch ein Mittel (18, 44), das fluiddurchlässig gemacht werden kann, getrennt ist, wobei besagtes Mittel derart positioniert ist, dass das Durchfliessen besagter reaktionsfähiger Komponente aus der besagten dritten Zone (15, 35) in die besagte zweite Zone (17, 33) vor dem Abschluss der Reaktion zwischen dem besagten, an eine feste Phase gebundenen Rezeptor und der nicht gebundenen Markierer tragenden Probe verhindert wird, und
(d) eine vierte Zone (19), welche in mindestens teilweisem Fluidkontakt mit besagter zweiter Zone (17, 35) steht und zur Fluidaufnahme aus besagter zweiter Zone (17, 33) angepasst ist.
(a) eine erste Zone (14, 32), welche eine Probe einer Vielzahl von unterschiedlichen, an eine feste Phase nicht gebundenen Rezeptoren enthält, die sich an besagte Komponente spezifisch binden, wobei einer der besagten Rezeptoren einen Markierer und ein zweiter keinen Markierer trägt,
(b) eine zweite Zone (17, 33), welche in mindestens teilweisem Fluidkontakt mit besagter erster Zone (14, 32) steht und zur Fluidaufnahme aus besagter erster Zone (14, 32) angepasst ist, wobei besagte zweite Zone (17, 33) einen an eine feste Phase gebundenen Rezeptor enthält, der sich an den keinen Markierer tragenden Rezeptor der ersten Zone bindet und sich mit besagtem markierten Rezeptor nicht spezifisch bindet,
(c) eine dritte Zone (15, 35), die eine reaktionsfähige Komponente enthält, welche reaktionsfähige Komponente sich mit dem Markierer auf besagter markierter Komponente, auf dem markierten Analogen der Komponente oder auf dem markierten Rezeptor unter Bildung eines detektierharen Komplexes verbindet, wobei besagte dritte Zone (15, 35) von besagter zweiter Zone (17, 33) durch ein Mittel (18, 44), das fluiddurchlässig gemacht werden kann, getrennt ist, wobei besagtes Mittel derart positioniert ist, dass das Durchfliessen besagter reaktionsfähiger Komponente aus der besagten dritten Zone (15, 35) in die besagte zweite Zone (17, 33) vor dem Abschluss der Reaktion zwischen dem besagten, an eine feste Phase gebundenen Rezeptor und der nicht gebundenen Markierer tragenden Probe verhindert wird, und
(d) eine vierte Zone (19), welche in mindestens teilweisem Fluidkontakt mit besagter zweiter Zone (17, 33) steht und zur Fluidaufnahme aus besagter zweiter Zone (17, 33) angepasst ist.
(a) eine erste Zone (14, 32), welche eine markierte Probe der (i) zu bestimmenden Komponente, (ii) eines Analogen der Komponente oder (iii) eines Rezeptors welcher sich an besagte Komponente spezifisch bindet, enthält,
(b) eine zweite Zone (17, 33), welche in mindestens teilweisem Fluidkontakt mit besagter erster Zone steht und zur Fluidaufnahme aus besagter erster Zone (14, 32) angepasst ist, wobei besagte zweite Zone (17, 33) einen an eine feste Phase gebundenen Rezeptor enthält, der sich an eines der folgenden bindet: (i) sowohl die Komponente der Probe als auch die markierte Komponente, (ii) sowohl die Komponente der Probe als auch das markierte Analoge der Komponente, oder (iii) den an die Komponente der Probe nicht gebundenen, markierten Rezeptor,
(c) eine dritte Zone (15, 35), die eine reaktionsfähige Komponente enthält, welche reaktionsfähige Komponente sich mit dem Markierer auf besagter markierter Komponente, auf dem markierten Analogen der Komponente oder auf dem markierten Rezeptor unter Bildung eines detektierbaren Komplexes verbindet, wobei besagte dritte Zone (15, 35) von besagter zweiter Zone (17, 33) durch eine Lücke oder einen Luftraum so getrennt ist, dass das Durchfliessen besagter reaktionsfähiger Komponente aus der besagten dritten Zone in die besagte zweite Zone vor dem Abschluss der Reaktion zwischen dem besagten, an die feste Phase gebundenen Rezeptor und der nicht gebundenen Markierer tragenden Probe verhindert wird, wobei sich besagte dritte Zone (15, 35) in Bezug auf besagte zweite Zone (17, 33) zwecks Schaffung von Fluidkontakt bewegt und dadurch das Durchfliessen der reaktionsfähigen Komponente in besagte zweite Zone in Gang setzt, und
(d) eine vierte Zone (19), welche in mindestens teilweisem Fluidkontakt mit besagter zweiter Zone (17, 33) steht und zur Fluidaufnahme aus besagter zweiter Zone (17, 33) angepasst ist.
(a) eine erste Zone (14, 32), welche eine Probe einer Vielzahl von unterschiedlichen, an eine feste Phase nicht gebundenen Rezeptoren enthält, die sich an besagte Komponente spezifisch binden, wobei einer der besagten Rezeptoren einen Markierer und ein zweiter keinen Markierer trägt,
(b) eine zweite Zone (17, 33), welche in mindestens teilweisem Fluidkontakt mit besagter erster Zone (14, 32) steht und zur Fluidaufnahme aus besagter erster Zone (14, 32) angepasst ist, wobei besagte zweite Zone (17, 33) einen an eine feste Phase gebundenen Rezeptor enthält, der sich an den keinen Markierer tragenden Rezeptor der ersten Zone bindet und sich mit besagtem markiertem Rezeptor nicht spezifisch bindet,
(c) eine dritte Zone (15, 35), welche eine reaktionsfähige Komponente enthält, welche reaktionsfähige Komponente sich mit dem Markierer auf besagter markierter Komponente, auf dem markierten Analogen der Komponente oder auf dem markierten Rezeptor unter Bildung eines detektierbaren Komplexesverbindet, wobei besagte dritte Zone (15, 35) von besagter zweiter Zone (17, 33) durch eine Lücke oder einen Luftraum so getrennt ist, dass das Durchfliessen besagter reaktionsfähiger Komponente aus der besagten dritten Zone (15, 35) in die besagte zweite Zone (17, 33) vor dem Abschluss der Reaktion zwischen dem besagten, an eine feste Phase gebundenen Rezeptor und der nicht gebundenen Markierer tragenden Probe verhindert wird, wobei sich besagte dritte Zone (15, 35) in Bezug auf besagte zweite Zone (17, 33) zur Schaffung von Fluidkontakt bewegt und dadurch das Durchfliessen der reaktionsfähigen Komponente in besagte zweite Zone (17, 33) in Gang setzt, und
(d) eine vierte Zone (19), welche in mindestens teilweisem Fluidkontakt mit besagter zweiter Zone (17, 33) steht und zur Fluidaufnahme aus besagter zweiter Zone (17, 33) angepasst ist.
(a) eine erste Zone (14, 32), welche eine markierte Probe der (i) zu bestimmenden Komponente, (ii) eines Analogen der Komponente oder (iii) einen Rezeptor, welcher sich an besagte Komponente spezifisch bindet, enthält,
(b) eine zweite Zone (42), welche in mindestens teilweisem Fluidkontakt mit besagter erster Zone steht und zur Fluidaufname aus besager erster Zone (14, 32) angepasst ist, wobei besagte zweite Zone (42) sowohl (1) einen an eine feste Phase gebundenen Rezeptor, der sich an eines der folgenden bindet: (a) sowohl die zu bestimmende Komponente als auch die markierte Komponente, (b) sowohl die Komponente als auch das markierte Analoge der Komponente, oder (c) den an die zu bestimmende Komponente nicht gebundenen, markierten Rezeptor, als auch (2) eine reaktionsfähige Komponente, welche reaktionsfähige Komponente sich mit besagtem Markierer unter Bildung eines detektierbaren Komplexes verbindet, und (3) Mittel zur Verzögerung der Verbindung des Markierers mit der reaktionsfähigen Komponente bis zum Abschluss der Reaktion zwischen dem an die feste Phase gebundenen Rezeptor und der nicht gebundenen Markierer tragenden Probe im wesentlichen abgeschlossen ist, enthält, und
(c) eine dritte Zone (19), welche in mindestens teilweisem Fluidkontakt mit besagter zweiter Zone (42) steht und zur Fluidaufnahme aus besagter zweiter Zone (42) angepasst ist.
(a) eine erste Zone (14, 32), welche eine Probe einer Vielzahl von unterschiedlichen, an eine feste Phase nicht gebundenen Rezeptoren enthält, die sich an besagte Komponente nicht spezifisch binden, wobei einer der besagten Rezeptoren einen Markierer und ein zweiter keinen Markierer trägt,
(b) eine zweite Zone (42), welche in mindestens teilweisem Fluidkontakt mit besagter erster Zone steht und zur Fluidaufnahme aus besagter erster Zone (14, 32) angepasst ist, wobei besagte zweite Zone sowohl (1) einen an eine feste Phase gebundenen Rezeptor, der sich an eines der folgenden bindet: (a) sowohl die zu bestimmende Komponente als auch die markierte Komponente, (b) sowohl die Komponente als auch das markierte Analoge der Komponente, oder (c) den an die zu bestimmende Komponente nicht gebundenen, markierten Rezeptor, als auch (2) eine reaktionsfähige Komponente, welche reaktionsfähige Komponente sich mit besagtem Markierer unter Bildung einer feststellbaren Entität verbindet, und (3) Mittel zur Verzögerung der Verbindung des Markierers mit der reaktionsfähigen Komponente bis die Reaktion des an die feste Phase gebundenen Rezeptors mit der nicht gebundenen Markierer tragenden Probe im wesentlichen abgeschlossen ist, enthält, und
(c) eine dritte Zone (19), welche in mindestens teilweisem Fluidkontakt mit besagter zweiter Zone (42) steht und zur Fluidaufnahme aus der zweiten Zone (42) angepasst ist.
(a) une première zone (14, 32) contenant un échantillon marqué d'un de (i) l'analyte à déterminer, (ii) un analogue de l'analyte et (iii) un récepteur qui se lie spécifiquement audit analyte,
(b) une seconde zone (17, 33) qui est en contact fluide au moins partiel avec ladite première zone et est adaptée pour recevoir un fluide provenant de ladite première zone (14, 32), ladite seconde zone (17, 33) contenant un récepteur de liaison en phase solide qui se lie à l'un parmi (i) à la fois l'analyte de l'échantillon et l'analyte marqué, (ii) à la fois l'analyte de l'échantillon et l'analogue d'analyte marqué ou (iii) le récepteur marqué non lié à l'analyte de l'échantillon,
(c) une troisième zone (15, 35) qui contient un composant réactif, composant réactif qui se combine avec la marque sur ledit analyte marqué, l'analogue d'analyte marqué ou le récepteur marqué afin de former une fraction détectable, ladite troisième zone (15, 35) étant séparée de ladite seconde zone (17, 33) par un moyen (18, 44) capable d'être rendu perméable au fluide, ledit moyen étant placé de façon à empêcher le passage dudit composant réactif provenant de ladite troisième zone (15, 35) et dans ladite seconde zone (17, 33) avant l'achèvement de la réaction entre ledit récepteur de liaison en phase solide et l'échantillon portant la marque non liée, et
(d) une quatrième zone (19) en contact fluide au moins partiel avec ladite seconde zone (17, 35) et adaptée pour recevoir un fluide provenant de ladite seconde zone (17, 33).
(a) une première zone (14, 32) contenant un échantillon d'un ensemble de récepteurs de liaison en phase non solide différents qui se lient spécifiquement audit analyte, un desdits récepteurs portant une marque et un second n'en portant pas,
b) une seconde zone (17, 33) qui est en contact fluide au moins partiel avec ladite première zone (14, 32) et est adaptée pour recevoir un fluide de ladite première zone (14, 32), ladite seconde zone (17, 33) contenant un récepteur en phase solide qui se lie au récepteur de la première zone qui ne porte pas de marque et n'est pas lié spécifiquement audit récepteur marqué,
(c) une troisième zone (15, 35) qui contient un composant réactif, composant réactif qui se combine avec la marque sur ledit analyte marqué, l'analogue d'analyte marqué ou le récepteur marqué afin de former une fraction détectable, ladite troisième zone (15, 35) étant séparée de ladite seconde zone (17, 33) par un moyen (18, 44) capable d'être rendu perméable au fluide, ledit moyen étant placé de façon à empêcher le passage dudit composant réactif de ladite troisième zone (15, 35) et dans ladite seconde zone (17, 33) avant l'achèvement de la réaction entre ledit récepteur de liaison en phase solide et l'échantillon portant la marque non liée, et
(d) une quatrième zone (19) en contact fluide au moins partiel avec ladite seconde zone (17, 33) et adaptée pour la réception d'un fluide provenant de ladite seconde zone (17, 33).
(a) une première zone (14, 32) contenant un échantillon marqué d'un de (i) l'analyte à déterminer, (ii) un analogue de l'analyte et (iii) un récepteur qui se lie spécifiquement audit analyte,
(b) une seconde zone (17, 33) qui est en contact fluide au moins partiel avec ladite première zone et est adaptée pour recevoir le fluide de ladite première zone (14, 32), ladite seconde zone (17, 33) contenant un récepteur de liaison en phase solide qui se lie à l'un parmi (i) à la fois l'analyte de l'échantillon et l'analyte marqué, (ii) à la fois l'analyte de l'échantillon et l'analogue d'analyte marqué, ou (iii) le récepteur marqué non lié à l'analyte de l'échantillon,
(c) une troisième zone (15, 35) qui contient un composant réactif, ledit composant réactif se combine avec la marque sur ledit analyte marqué, l'analogue d'analyte marqué ou le récepteur marqué afin de former une fraction détectable, ladite troisième zone (15, 35) étant séparée de ladite seconde zone (17, 33) par un intervalle ou un espace d'air de façon à empêcher le passage dudit composant réactif de ladite troisième zone dans ladite seconde zone avant l'achèvement de la réaction entre ledit récepteur de liaison en phase solide et l'échantillon portant la marque non liée, dans lequel ladite troisième zone (15, 35) se déplace par rapport à ladite seconde zone (17, 33) afin de créer un contact fluide et amorcer ainsi le transfert du composant réactif vers ladite seconde zone, et
(d) une quatrième zone (19) en contact fluide au moins partiel avec ladite seconde zone (17, 35) et adaptée pour recevoir le fluide provenant de ladite seconde zone (17, 33).
(a) une première zone (14, 32) contenant un échantillon d'un ensemble de récepteurs de liaison en phase non solide différents qui se lient spécifiquement audit analyte, un desdits récepteurs portant une marque et un second n'en portant pas,
b) une seconde zone (17, 33) qui est en contact fluide au moins partiel avec ladite première zone (14, 32) et est adaptée pour recevoir un fluide provenant de ladite première zone (14, 32), ladite seconde zone (17, 33) contenant un récepteur en phase solide qui se lie au récepteur de première zone qui ne porte pas de marque et non lié audit récepteur marqué,
(c) une troisième zone (15, 35) qui contient un composant réactif, composant réactif qui se combine avec la marque sur ledit analyte marqué, l'analogue d'analyte marqué ou le récepteur marqué afin de former une fraction détectable, ladite troisième zone (15, 35) étant séparée de ladite seconde zone (17, 33) par un intervalle ou un espace d'air de façon à empêcher le passage dudit composant réactif de ladite troisième zone (15, 35) dans ladite seconde zone (17, 33) avant l'achèvement de la réaction entre ledit récepteur de liaison en phase solide et l'échantillon portant une marque non liée, dans lequel ladite troisième zone (15, 35) se déplace par rapport à ladite seconde zone (17, 33) afin de créer un contact fluide et d'amorcer ainsi un transfert du composant réactif vers ladite seconde zone (17, 33)
(d) une quatrième zone (19) en contact fluide au moins partiel avec ladite seconde zone (17, 33) et adaptée pour la réception un fluide provenant de ladite seconde zone (17, 33).
(a) une première zone (14, 32) contenant un échantillon marqué d'un de (i) l'analyte à déterminer, (ii) un analogue de l'analyte et (iii) un récepteur qui de lie spécifiquement audit analyte,
(b) une seconde zone (42) qui est en contact fluide au moins partiel avec ladite première zone et adaptée pour recevoir un fluide provenant de ladite première zone (14, 32), ladite seconde zone (42) contenant à la fois (1) un récepteur de liaison en phase solide qui se lie à un de a) à la fois l'analyte à déterminer et l'analyte marqué, (b) à la fois l'analyte et l'analogue d'analyte marqué, ou (c) le récepteur marqué qui n'est pas lié à l'analyte à déterminer, (2) un composant réactif, ledit composant réactif se combine avec ladite marque afin de former une fraction détectable et (3) un moyen pour retarder la combinaison de la marque et du composant réactif jusqu'à ce que la réaction du récepteur de liaison en phase solide avec l'échantillon portant la marque non liée soit pratiquement complète, et
(c) une troisième zone (19) en contact fluide au moins partiel avec ladite seconde zone (42) et adaptée pour recevoir un fluide provenant de ladite seconde zone (42).
(a) une première zone (14, 32) contenant un échantillon d'un ensemble de récepteurs de liaison en phase non solide différents qui se lient spécifiquement audit analyte, un desdits récepteurs portant une marque et un second n'en portant pas,
b) une seconde zone (42) qui est en contact fluide au moins partiel avec ladite première zone et qui est adaptée pour recevoir un fluide provenant de ladite première zone (14, 32), ladite seconde zone contenant à la fois un récepteur en phase solide qui se lie à un de (a) à la fois l'analyte à déterminer et l'analyte marqué, (b) à la fois l'analyte et l'analogue d'analyte marqué ou (c) le récepteur marqué qui n'est pas lié à l'analyte à déterminer, (2) un composant réactif, ledit composant réactif se combine avec ladite marque afin de former une fraction détectable et (3) un moyen pour retarder la combinaison de la marque et du composant réactif jusqu'à ce que la réaction du récepteur de liaison en phase solide avec l'échantillon portant la marque non liée soit pratiquement achevée, et
(c) une troisième zone (19) en contact fluide au moins partiel avec ladite seconde zone (42) et adaptée pour recevoir un fluide provenant de ladite seconde zone (42).